The present invention, in some embodiments thereof, relates to methods and kits for analyzing DNA binding moieties attached to DNA, and, more particularly, but not exclusively for analyzing histone binding in a cell.
Cell type specific functions and response rely on tight gene regulation, orchestrated by the chromatin landscape. The current dogma of cellular differentiation set forth more than half a century ago by Conrad Waddington presents a model of a progressive closing of the genome. According to this model, differentiation is a gradual transition from an open chromatin state in multipotent stem cells to a compacted chromatin state in differentiated cells. This model is supported by recent genome-wide histone modification analyzing presence or level of embryonic stem cells compared with terminally differentiated cells. Hematopoiesis is a paradigmatic differentiation process where a single hematopoietic stem cell gives rise to a large number of cell types (essentially the entire blood system) through a series of characterized intermediate progenitor cells. Chromatin regulation has a central role in hematopoiesis and mutations or loss of chromatin factors critically alter the hematopoietic outcome. Moreover, genome-wide chromatin analyzing presence or level of studies have revealed big differences in the histone modifications and TF binding maps in different mature immune cells. All these findings point to dynamic chromatin rearrangements at some point during hematopoiesis.
Comprehensive study of the chromatin events during hematopoiesis has been hampered by the low sensitivity and reproducibility for small cell numbers with current chromatin immunoprecipitation (ChIP) protocols. These protocols require several enzymatic steps with limited performance when the input DNA is below the nanogram range. While an average diploid mammalian cell has roughly 4-8 pg of DNA, losses following ChIP reduces the available DNA for analysis by 2-3 orders of magnitude, setting the lower limit for genome-wide chromatin analysis at 50,000 cells (T. S. Furey, Nat. Rev. Genet. 13, 840-852 (2012)).
Amplification of ChIP material partially alleviate this problem at the cost of introducing amplification biases (P. Shankaranarayanan et al., Nat. Methods 8, 565-567 (2011); M. Garber et al., Mol Cell 47, 810-822 (2012). In addition, such amplification processes, make ChIP protocols laborious and bias prone.
Additional background art includes Blecher Gonen et al., Nature Protocols, 8, 539-554 (2013), US Patent Application No. 20140024052, WO 2013134261 and WO 2002014550.